Solid-phase peptide synthesis (SPPS) involves a covalent attachment step (i.e., anchoring) that links the nascent peptide chain to an insoluble polymeric support (i.e., support material) containing appropriate functional groups for attachment. Subsequently, the anchored peptide is extended by a series of addition (deprotection/coupling) cycles that involve adding N.sup..alpha. -protected and side-chain-protected amino acids stepwise in the C to N direction. Once chain assembly has been accomplished, protecting groups are removed and the peptide is cleaved from the support.
Typically, SPPS begins by using a handle to attach the initial amino acid residue to the functionalized polymeric support. A handle (i.e., linker) is a bifunctional spacer that, on one end, incorporates features of a smoothly cleavable protecting group, and on the other end, a functional group, often a carboxyl group, that can be activated to allow coupling to the functionalized polymeric support. Known handles include acid-labile p-alkoxybenzyl (PAB) handles, photolabile o-nitrobenzyl ester handles, and handles such as those described by Albericio et al., J. Org. Chem., 55, 3730-3743 (1990) and references cited therein, and in U.S. Pat. Nos. 5,117,009 (Barany) and 5,196,566 (Barany et al.).
For example, if the support material is prepared with amino-functionalized monomers, typically, the appropriate handles are coupled quantitatively in a single step onto the amino-functionalized supports to provide a general starting point of well-defined structures for peptide chain assembly. The handle protecting group is removed and the C-terminal residue of the N.sup..alpha. -protected first amino acid is coupled quantitatively to the handle. Once the handle is coupled to the solid-phase and the initial amino acid or peptide is attached to the handle, the general synthesis cycle proceeds. The synthesis cycle generally consists of deprotection of the N.sup..alpha. -amino group of the amino acid or peptide on the resin, washing, and, if necessary, a neutralization step, followed by reaction with a carboxyl-activated form of the next N.sup..alpha. -protected amino acid. The cycle is repeated to form the peptide or protein of interest. Solid-phase peptide synthesis methods using functionalized insoluble supports are well known. See, for example, Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Barany and Merrifield, In The Peptides, Vol. 2, pp. 1-284 (1979); Gross, E. and Meienhofer, J., Eds., Academic: New York; and Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987).
Most current methods of SPPS rely on the .alpha.-carboxyl function of the eventual C-terminal amino acid residue to achieve anchoring to the support. However, this approach limits SPPS to the formation of peptides having acid, amide, or monosubstituted amide functionality, for example, as the C-terminal functionality, unless more complex procedures are used. Furthermore, certain functionalities, such as aldehydes, cannot typically be obtained using this approach. Cyclic peptides are also not possible using this method. Also, racemization of sensitive amino acid residues in the synthesis of peptide acids is a problem using this method.
Side-chain anchoring, i.e., methods of SPPS that use amino acids with side-chain functional groups for attachment of peptides, is potentially useful for the formation of unusual C-terminal functionalities as well as cyclic peptides. However, side-chain anchoring is inherently limited to certain trifunctional amino acids. Therefore, it would be desirable to develop a general method of SPPS that: (1) allows for the preparation of a wider variety of peptides; (2) does not typically result in racemization of sensitive amino acid residues; and (3) can incorporate a wider variety of amino acids into cyclic peptides.